Oranges and Human Health

The orange is a widely consumed fruit, playing a central role in both food security and public health. Its broad acceptance worldwide stems from its pleasant taste and rich nutritional profile, making it a symbol of healthy eating. A single medium orange provides nearly 100% of the recommended daily intake of vitamin C ^[1,3].

The biological importance of vitamin C is associated with different metabolic processes in our body, such as collagen synthesis, iron absorption, and the strengthening of the immune system. Other important bioactive compounds found in oranges include the flavonoids hesperidin, naringenin, and diosmin. These compounds exert significant anti-inflammatory, antioxidant, and cardioprotective effects. Furthermore, carotenoids such as beta-cryptoxanthin, lutein, and zeaxanthin play a fundamental role in protecting vision and supporting the immune system ^[3,4].

In addition, the presence of dietary fibers in citrus fruits, especially pectins, contributes to the reduction of LDL cholesterol, improves intestinal transit, and acts as prebiotics by stimulating the growth of beneficial bacteria in the gut, such as Lactobacillus sp. and Bifidobacterium sp. Other nutrients present in oranges include potassium, calcium, folic acid, and thiamine, making this fruit a complete and functional food ^[5,7,8,9].

Citrus Production

Beyond their nutritional value, oranges serve as raw material for several industrial processes, including essential oil extraction for the pharmaceutical industry and pulp separation for animal feed. However, the most important use is for orange juice production, being the fruit’s main commercial application and a staple for direct human consumption ^[10].

In the 2024/25 harvest alone, global orange production reached 45.22 million tons, with the largest producers being Brazil, China, and the European Union. The citrus value chain generates billions of dollars in exports and millions of direct and indirect jobs worldwide, making it a strategic sector for multiple economies, especially for Brazil ^{[11,13,16,32]}. Our country holds an undisputed leadership in the global citrus market, being the largest producer of orange juice, representing 70% of the global market (1.01 million metric tons in the 2024/25 season) and 29% of the world’s orange supply (13 million metric tons) ^[11,12].

Thus, it is evident that orange and orange juice production stand out in the Brazilian economy, generating approximately US$3.48 billion from jobs and commercialized products. This data highlights the strategic importance of the citrus sector at both nutritional and socioeconomic levels ^{[11, 16]}.

Threats

Despite its global leadership, Brazil’s citrus industry faces critical challenges. Historical data reveal a consistent decline in productivity: between 1994/95 and 2003/04, the average output was 351 million orange boxes (each box weighing 40.8kg); it dropped to 337 million boxes in the following decade and 308 million between 2014/15 and 2023/24. Consequently, many citrus growers find themselves in a vulnerable position, awaiting the development of new technological tools to combat these diseases. ^{[14,17,18,19,32]}.

Worldwide, the most destructive challenge in citrus farms is Greening, caused by a non-cultivable bacterium Candidatus Liberibacter spp., which is transmitted by the Asian citrus psyllid. Globally, Greening has already led to the destruction of more than 60 million citrus trees, resulting in collapsed production in entire regions, including India, Indonesia, Africa, and Florida (USA). In Brazil, the fruit drop rate caused solely by Greening was responsible for a production drop of approximately 25% in the 2024/25 harvest, corresponding to a loss of approximately 3.115 million tons of oranges ^{[14, 20,21, 22]}.

Furthermore, around one-third of global fruit and vegetable production is lost along the supply chain, from harvest to transportation and storage. The citrus market is no exception and is also highly vulnerable to diseases that severely impact production, such as post-harvest pathogens. ^[15]

The fungus Penicillium digitatum, responsible for Green mold, accounts for up to 90% of fruit losses, rapidly deteriorating oranges, and drastically reducing shelf life. In a meeting with AlfaCitrus, we learned firsthand about their concerns and problems with controlling Geotrichum candidum (Sour Rot), another pathogen responsible for productivity losses for which no commercial fungicide exists. Sour rot is responsible for substantial losses, especially in high-rainfall seasons. Together, these diseases cause nearly 50% of losses of all oranges produced, compromising fruit availability in a short time ^{[25, 26, 27]}.

Additionally, in the absence of effective control for these diseases, the current management relies on the intensive use of insecticides and fungicides. According to the Food and Agriculture Organization of the United Nations (FAO), global agriculture used 3.5 million tons of agrochemicals in 2021, twice the volume reported in 1991, demonstrating a steady upward trend in pesticide consumption. In the same year, Brazil applied 719,500 tons, ranking as the world's largest consumer of pesticides^[27,28,29].

Especially in citrus production, according to the Pesticide Action Network (PAN), oranges are among the 12 most contaminated foods with agrochemicals, with substances classified as Highly Hazardous Pesticides (HHPs) by the UN ^[6].

From an environmental perspective, the lack of specificity of pesticides leads to the intoxication of non-target organisms. As a result, the biological processes of these living beings are compromised, disrupting the ecological balance. Also, the indiscriminate use of antibiotics against Greening raises concerns about the potential emergence of resistant strains. Moreover, these approaches face critical limitations: only partial effectiveness, environmental risks, stricter regulatory restrictions, and escalating production costs that undermine profitability ^[6,28,29].

As a reflection of the decline in productivity and fruit loss in the production chain, the price of a 48 kg orange box more than doubled, rising 130% from US 9.92 to US 22.86 dollars. This sharp increase has directly contributed to a decline in the consumption of oranges and their derivatives, limiting access to their nutritional benefits (Human Practices). Consequently, food security is undermined both in Brazil and worldwide, as reduced intake of the fruit exacerbates micronutrient deficiencies and compromises dietary quality ^{[22, 23, 33, 34, 35]}.

Our Solution: Pepcitrus!

This desperate scenario creates a structural global challenge that impacts not only stakeholders such as farmers and industries but also to consumers and entire communities that depend on this economy. After an extensive literature review and different conversations about alternative strategies, we identified antimicrobial peptides (AMPs) as a promising alternative solution for conventional phytopathology management.

Thus, we reviewed previous iGEM projects exploring different applications of AMPs and strategies against citrus pathogens. Going beyond previous approaches, we set out to pioneer the use of an AMP that had never been tested against the major citrus diseases ^[31].

We reached out to the FSU and Montpellier teams to organize a meeting and discuss project conceptualization around using peptides. In particular, FSU provided us with insights regarding Clas.

Led by conversations with professors and experts in the field, we discovered CTX, an AMP isolated from the Brazilian Cerrado frog Hypsiboas albopunctatus. In the case of CTX, the primary mechanism of action involves disrupting the plasma membrane through pore formation, which leads to cytoplasmic leakage and subsequent cell death ^[30].

In that way, our goal is to evaluate the efficacy of applying CTX to combat the most devastating pre- and post-harvest citrus diseases: Greening, Green Mold and Sour Rot. To turn this vision into reality and save the oranges, we defined a clear set of goals:

1. Test CTX against the major citrus pathogens: Candidatus Liberibacter spp. (Greening), Penicillium digitatum (Green Mold), Geotrichum candidum (Sour Rot).
2. Produce CTX through different hosts using genetically engineered Escherichia coli, Saccharomyces cerevisiae, and Aspergillus oryzae. Assess production efficiency across hosts and cultivation methods (liquid and solid-state fermentation).
3. Check the possibility of applying CTX with a scalable production strategy that ensures high efficiency, low cost, and sustainability through a circular economy framework. Thus, aiming to make it possible real life applications in oranges and trees.

Our first objective is to test the efficiency of CTX against the major citrus diseases: Greening (Candidatus Liberibacter spp.), Green Mold (Penicillium digitatum), and Sour Rot (Geotrichum candidum). To make this possible, we built an extensive network of collaborations with laboratories, researchers, and stakeholders specializing in each of these target diseases.

In the case of Greening, since Candidatus Liberibacter spp. cannot be cultivated under laboratory conditions, we designed an alternative experiment by applying CTX directly to infected citrus leaves, and assessed its effectiveness through RT-qPCR analysis.

We realized that conventional RT-qPCR analysis is costly, time-consuming, and requires specialized laboratory infrastructure. To address it, we developed a hardware device capable of rapid detection of Greening, inspired by the remarkable ability of dogs to detect disease. Our innovative hardware design integrates an electronic nose (eNose) with machine learning algorithms to identify infected leaves quickly. This will enable large-scale disease detection and application, while also facilitating the development of treatments for Greening by reducing diagnostic time and increasing detection accuracy.

Our team went further, recognizing the complexity of Greening as a phloem-restricted disease and the need to overcome the challenges of developing laboratory assays that accurately capture plant-based dynamics. To this end, we developed a mathematical model. This model elucidated key features of the disease, including bacterial movement within the phloem and the plant’s immune response, allowing us to better understand disease dynamics through simulation of its progression. Leveraging these insights, we established a framework to optimize CTX dosage and application schedules, which is critical for large-scale deployment.

In this way, considering the widespread impact of pre- and post-harvest diseases, we recognized that meeting the demands of this massive agricultural market would require producing CTX at a large scale and in significant quantities. Only considering Greening in our region, treating the disease would require approximately 3 tons per year. Even when considering only this regional pre-harvest market, the potential demand for antimicrobial peptides like CTX becomes clear.

This is where synthetic biology came in, with a strategy to produce CTX through bioprocess strategies with genetically engineered microorganisms. However, producing AMPs in microorganisms is a major challenge, as these peptides are often toxic to the host itself. To overcome this, we applied the Design–Build–Test–Learn (DBTL) cycle and developed a novel strategy: fusing the CTX to highly expressed carrier proteins and cleaving it afterward.

To assess production efficiency, we evaluated three different hosts, Escherichia coli, Saccharomyces cerevisiae, and Aspergillus oryzae, and compared the Titer, Rate, and Yield (TRY) of each system, ultimately identifying the most cost-effective strategy for large-scale CTX production.

A key highlight of our approach was leveraging the high protein secretion capacity of A. oryzae. To this end, we tested CTX production by fusing it to the organism’s most abundantly expressed protein, taking advantage of its natural secretion system. To advance toward a scalable production within a circular economy framework, we explored solid-state fermentation with A. oryzae using orange peel residues as a substrate for CTX production. Thus establishing a circular and environmentally responsible model for peptide manufacturing ensuring high efficiency, low cost, and sustainability.

In line with the principles of open science, we successfully registered CTX (Bba_250NI4BC) and sfGFP-CTX (BBa_25SVFSZB) as new biological and composite parts in the iGEM Registry. This opens the door for future iGEM teams to explore the full potential of CTX across a wide range of applications.

Our journey brought together a complex and interdisciplinary network, driven by a strong Human Practices component and comprising more than 90 specialists and stakeholders, integrating different and interdisciplinary visions. Throughout this wiki, you will find details of our experiments, engineering strategies, modeling approaches, economics analysis, sustainability development, and hardware design, all of which are intertwined with multiple layers of society.

We invite you to explore the project outcomes and see how our work not only offers a new tool against devastating citrus diseases but also opens the door to scalable peptide production for agricultural applications. In this way, Pepcitrus aims to build a more sustainable future for citrus farming while bringing joy and orange security to people's tables. Together, we're not just saving oranges, but protecting livelihoods, boosting sustainability and shaping a healthier future for all.